The invention is concerned with methods for use in the identification of compounds which affect the activity of a physiologically important calcium pump, the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA).
In most animal cells and plant cells, the normal concentration of free cytosolic Ca2+ is 50 to 100 nM. Since Ca2+ acts as a major intracellular messenger, elevating these levels affects a wide range of cellular processes including contraction, secretion and cell cycling (Dawson, 1990, Essays Biochem. 25:1-37; Evans et al., 1991, J. Exp. Botany 42:285-303). Intracellular Ca2+ stores hold a key position in the intracellular signalling. They allow the rapid establishment of Ca2+ gradients, and accumulate and release Ca2+ in order to control cytosolic Ca2+ levels. Moreover, lumenal Ca2+ intervenes in the regulation of the synthesis, folding and sorting of proteins in the endoplasmic reticulum (Brostrom and Brostrom, 1990, Ann. Rev. Physiol. 52:577-590; Suzuki et al., 1991, J. Cell. Biol. 114:189-205; Wileman et al., 1991, J. Biol. Chem. 266:4500-4507). Furthermore it controls signal-mediated and passive diffusion through the nuclear pore (Greber and Gerace, 1995, J. Cell. Biol. 128:5-14).
Three genes that code for five different isoforms of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) are known in vertebrates, SERCA1a/b, SERCA2a/b and SERCA3. The SERCA isoforms are usually tagged to the endoplasmic reticulum (ER) or ER subdomains like the sarcoplasmic reticulum, although the precise subcellular location is often not known. The SERCA proteins belong to the group of ATP-driven ion-motive ATPases, which also includes, amongst others, the plasma membrane Ca2+-transport ATPases (PMCA), the Na+-K+-ATPases, and the gastric H+-K+-ATPases. The SERCA Ca2+-transport ATPases can be distinguished from their plasma membrane counterparts like PMCA by the specific SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)-1,4-benzohydroquinone (Thastrup et al., 1990, PNAS 87:2466-2477; Seidler et al., 1989, J. Biol. Chem. 264:17816-17823; Oldershaw and Taylor, 1990, FEBS Lett. 274:214-216). In view of the diverse role of Ca2+ in the cell and the fact that Ca2+ is stored in diverse organelles, the diversity in Ca2+-accumulation pump isoforms is not surprising.
SERCA1 is only expressed in fast-twitch skeletal muscle fibres. The gene encodes two different isoforms; SERCA1b which is the neonatal isoform and SERCA1a the adult isoform (Brandl et al., 1986, Cell 44:597-607; Brandl et al., 1987, J. Biol. Chem. 262:3768-3774). The difference between the two isoforms is the result of an alternative splice. As a consequence, the neonatal isoform contains a highly charged carboxyl-terminal extension (Korczak et al., 1988, J. Biol. Chem. 263:4813-4819). The reason for this alternative splicing is as yet unknown; the functional significance of this extension is not yet clear. When expressed in COS cells, SERCA1a and SERCA1b exhibit nearly identical maximal Ca2+-turnover rate, Ca2+-affinity and ATP-dependency of Ca2+ transport (Maruyama and MacLennan, 1988, PNAS 85:3314-3318). The human SERCA1 gene is mapped on chromosome 16P12.1 and is about 26 kb long (MacLennan et al., 1987, Somatic Cell Mol. Genet. 13:341-346; Callen et al., 1991, Am. J. Hum. Genet. 49:1372-1377).
SERCA2 is expressed in muscle and non-muscle cells. The human SERCA2 gene maps to chromosome 12q23-q24.1 (Otsu et al., 1993, Genomics 17:507-509). Partial sequence analysis suggests that the same exon/intron layout is conserved between SERCA1 and SERCA2. mRNA of SERCA2 can be divided in 4 different classes; class 1 encodes SERCA2a and is mainly expressed in muscle, the other classes encode SERCA2b and are mainly expressed in non-muscle tissues. SERCA2b harbors a 49 amino acid extension, which contains a highly hydrophobic stretch. As with SERCA1, no functional difference can be measured between the two SERCA2 isoforms when expressed in COS cells (Campbell, 1991, J. Biol. Chem. 266:16050-16055). However, differences in Ca2+ affinity and turnover rate of the phosphoprotein intermediate have been observed (Lytton et al., 1992, 267:14483-14489; Verboomen et al., 1992, J. Biochem. 286:591-596). Both isoforms are expressed in a tissue-dependent pattern, both qualitatively and quantitatively (Eggermont et al., 1990, J. Biochem. 271:649-653). Cardiac muscle expresses 5- to 20-fold higher levels of SERCA2 than smooth muscle. Slow-twitch skeletal and cardiac muscle only express SERCA2a, while SERCA2b (referred to as the xe2x80x9chousekeepingxe2x80x9d isoform) is expressed in all non-muscle tissue, and represents about 75% of the Ca2+-transporting ATPase activity in smooth-muscle tissue. Different protein-to-message ratios for SERCA2a and SERCA2b have been observed. Cardiac muscle expresses 70 times more protein and only 7 times more SERCA2a mRNA compared to stomach smooth muscle which expresses SERCA2b (Khan et al., 1990, J. Biochem. 268:415-419).
SERCA3 is considered to be the non-muscle SERCA isoform. SERCA3 lacks the putative interacting domain for phospholamban, and hence, does not respond to this modulator (Toyofuku et al., 1993, J. Biol. Chem. 268:2809-2815). When expressed in COS cells, SERCA3 shows approximately 5-fold lower activity for Ca2+ and a slightly higher pH optimum (Toyofuku et al., 1992, J. Biol. Chem. 267:14490-14496). In platelets, mast cells and lymphoid cells SERCA3 is co-expressed with SERCA2b (Wuytack et al., 1994, J. Biol. Chem. 269:1410-1416; Wuytack et al., 1995, Bioscience Rep. 15:299-306). Expression has also been observed in some arterial endothelial cells, in early developing rat heart, in some secretory epithelial cells of endodermal origin and in cerebellar Purkinje neurons.
In slow-twitch skeletal muscle, cardiac muscle and smooth-muscle tissues, SERCA2 activity is modulated by phosphorylation of the regulatory protein phospholamban (PLB) (see Fuji et al., 1991, FEBS Lett. 273:232-234). In cardiac muscle, in vivo phosphorylation of PLB by cAMP- or Ca2+/Calmodulin-dependent protein kinase has a positive effect on the Ca2+ transport (Le Peuch et al., 1997, Biochemistry 18:5150-5157; Tada et al., 1979, J. Biol. Chem. 254: 319-326; Davis et al., 1983, J. Biol. Chem. 258:13587-13591; Wegener et al., 1989, J. Biol. Chem. 264:11468-11474). In order to determine the exact in vivo role of phospholamban, PLB-deficient mice have been generated (Luo et al., 1994, Circ. Res. 75:401-409). A marked effect is observed on Ca2+ uptake, whereas no effect is measured in Vmax. The ablation of the PLB gene in mice is associated with increased myocardial contractility, and a loss of the positive inotropic response to adrenergic stimulation. The precise molecular mechanism underlying the modulation of SERCA by PLB is not apparent. An electrostatic mechanism has been proposed, as a direct interaction between PLB and SERCA, in which the unphosphorylated PLB inhibits the SERCA pump (Kirchberger et al., 1986, Biochemistry 25:5484-5492; Chiesi and Schwaller, 1989, FEBS Lett. 244:241-244; Xu and Kirchberger, 1989, J. Biol. Chem. 264:16644-16651). Alternatively, PLB and the SERCA Ca2+ pump are able to interact and phosphorylation of PLB alters its properties, as confirmed by cross-linking experiments (James et al., 1989, Nature 342:90-92). In some experiments, inhibitory effects of PLB have been observed on co-transfection of PLB and SERCA2a in COS-1 cells (Fuji et al. 1990, FEBS Lett. 273:232-234). Several models have been proposed to explain the regulatory effect of PLB on Ca2+ ATPases. These include the aggregation of SERCA2 around a pentameric form of PLB (Voss et al., 1994, Biophys J. 67:190-196). Another explanation starts from the electrostatic inhibition of Ca2+ binding due to the SERCA-PLB interaction (Toyoftiku et al., 1994, J. Biol. Chem. 269:3088-3094). The interaction between PLB and SERCA2a has been studied in more detail, revealing a putative PLB binding domain that is also present SERCA1 but not in SERCA3. This has been further confirmed by expression studies in COS-1 cells (Toyofuku et al., 1993, J. Biol. Chem., 268:2809-2815). Such a finding is remarkable as SERCA1 and PLB are never co-expressed in vivo.
Direct phosphorylation of SERCA by Ca2+/CaM kinase II results in a 2-fold higher maximal velocity Xu and Kirchberger, 1989, J. Biol. Chem. 264: 16644-16651. This CaM kinase phosphorylation is specific for SERCA2 and may act synergistically with the phosphorylation of phospholamban.
Sarcolipin (SLN) is a peptide of 33 amino acids in length that co-purifies with SERCA1. The human gene encoding SLN was mapped to chromosome 11q22-q23. The protein sequence shows some homology to phospholamban, especially in the lumenal part of the protein. In rabbits SLN is highly expressed in fast-twitch skeletal muscle, as is SERCA1 (Odermatt et al., 1997, Genomics 45:741-553). In co-expression studies in HEK-293 T-cells, a decrease of SERCA1 affinity for Ca2+ was observed, but maximal Ca2+ uptake rates were stimulated. Mutational analysis provided evidence for different mechanisms of interaction of both SLN and PLB with the SERCA molecules (Odermatt, et al., 1998, J. Biol. Chem. 273:12360-12369).
SERCA plays an important role in regulating Ca2+ levels, and hence in pathologies related to abnormal Ca2+ concentrations and regulation. For instance, abnormal cytosolic free Ca2+ levels are involved in different muscle pathologies (Morgan, 1991, N. Engl. J. Med. 325:625-632; Perreault et al., 1993, Circulation 87 Suppl. VII:31-37). Other major pathologies in which SERCA may play a role include cardiac hypertrophy, heart failure, and hypertension (Arai et al., 1994, Circ. Res. 74:555-564; Lompre et al., 1994, J. Mol. Cell. Cardiol. 26:1109-1121).
Cardiac hypertrophy is an adaptive response of the cardiac muscle to a hemodynamic overload, in which diastolic dysfunction is one of the earliest signs of pathological hypertrophic response. In animal models, where most studies are performed, a highly significant positive correlation has been obtained between end-diastolic cytosolic Ca2+ levels and diastolic relaxation abnormalities. After aortic binding, SERCA2 mRNA and protein levels are decreased, as is the sarcoplasmic reticulum Ca2+ uptake (Komuro et al., 1989, J. Clin Invest. 83:1102-1108; de la Bastie et al., 1990, Circ. Res. 66:554-564). This effect was only found in cases of severe hypertrophy, and was only observed when heart failure occurs. In moderate hypertropy and in cases of compensated hypertrophy no changes in the level of SERCA mRNA were observed (de la Bastie et al, ibid; Feldman et al., 1993, Circ. Res. 73:184-192).
In humans, most studies report a decrease in SERCA2 mRNA, SERCA2 protein levels and decreased Ca2+ uptake in a failing heart (Arai et al., 1993, Circ. Res. 72:463-469; Hasenfuss et al., 1994, Circ. Res., 75: 434-442). The decreased levels of SERCA2 expression are accompanied by decreased expression of phospholamban, cardiac ryanodine receptor and dihydropyridine receptor (Vatner et al., 1994, Circulation 90:1423-1430; Go et al., 1995, J. Clin. Invest. 95:888-894; Takahashi et al., 1992, J. Clin. Invest. 90:927-935). These human heart failure data are confirmed in different animal models. In a hypertrophic animals, SERCA2 expression levels are decreased; in a dilated strain Ca2+ uptake is decreased with increasing age (Kuo et al., 1992, Biochem Biophys acta 1138:343-349; Whitmer et al., 1988, Circ. Res. 62:81-85). Most striking in both humans and animal models is the strong positive correlation between SERCA2 and phospholamban mRNA levels. Examples in literature that do not confirm these data are most likely the result of various pathogenic mechanisms that can lead to heart failure.
Blood vessels from hypertensive animals have an increased wall thickness and show altered contractile properties. Several lines of evidence indicate that diminished Ca2+ pump activities might contribute to elevation in cytoplasmic Ca2+ levels in hypertension. However, increased expression of SERCA2 has also been observed. Further study is required to resolve these contradictory results.
Darier-White disease is an autosomal-dominant skin disorder characterized by loss of adhesion between epidermal cells (acantholysis) and abnormal keratinization. In several patients mutations have been found in SERCA2, demonstrating the role of SERCA and Ca2+-signalling pathway in the regulation of cell-to-cell adhesion and differentiation of the epidermis (Sakuntabhai et al., 1999, Nature Genetics 21:271-277).
Although little is known about the involvement of SERCA in skeletal muscle disorders, deficiency in the Ca2+-transport ATPase activity has been found in Brodys disease (Benders et al., 1994, J. Clin. Res. 94:741-748). The disorder is characterized by exercise-induced impairment of muscle relaxation. Normal levels of SERCA1 protein were detected, but the SERCA activity was decreased by about 50% in patients suffering from the disease. In other research, SERCA1 in fast-twitch fibers of Brody patients could not be detected immunologically (Danon et al., 1988, Neurology 38:812-815). However, three Brody patients show no defects in their SERCA1 gene, indicating pleiotropic mechanisms underlying Brody disease (Zhang et al., 1995, Genomics 30:415-424).
The underlying mechanism of non-insulin-dependent diabetes mellitus (NIDDM) is still unknown. In islets of Lagerhans from db/db mice (a NIDDM model), glucose-induced initial induction and subsequent oscillations of intracellular Ca2+ concentrations were absent. Further analysis showed that SERCA3 was almost entirely lacking from the db/db islets. These results and thapsigargin experiments implicate SERCA3 in the defective insulin secretion associated with NIDDM (Roe et al., 1994, J. Biol. Chem. 269:18279-28282). A significant reduction of SERCA3 expression was also found in Goto-Kakizaki rats, a non-obese model of NIDDM (Varadi et al., 1996, J. Biochem. 319:521-527) Interactions have been reported between different SERCAs (SERCA1 and SERCA2) and different Insulin Receptor Substrates (IRS-1 and IRS-2). This interaction was dependant on insulin (Algenstaedt et al., 1997, J. Biol. Chem. 272:23696-23702). Inactivation of IRS-2 has recently been shown to resemble certain aspects of type 2 diabetes (Withers et al., 1998, Nature 391:900-904).
In mammals, there are three genes encoding different SERCA isoforms. In contrast, the nematode worm Caenorhabditis elegans (C. elegans) has only a single homologue of the mammalian SERCA protein, which was identified by the C. elegans genome-sequencing consortium (see Science issue 282, 1998). The C. elegans SERCA gene is located on chromosome III on a cosmid named K11D9. On a physical level, the gene consists of six exons that span an Open Reading Frame of 3.2 kb, resulting in a predicted protein of 1059 amino acids. The consensus alternative splice site that is present in the C-terminal end of mammalian SERCA genes is present in the worm as well. This leads to a second isoform consisting of 7 exons that span an ORF of 3.0 kb, resulting in a protein of 1004 amino acids. This may indicate a functional conservation of this domain of the protein, e.g. in regulating the activity of the SERCA pump.
C. elegans is a small roundworm that has a life span of only three days, allowing rapid accumulation of large quantities of individual worms. The cell-lineage is fixed, allowing identification of each cell which has the same position and developmental potential in each individual animal. C. elegans is extremely amenable to genetic approaches and a large collection of mutants have been isolated that are defective in embryonic development, behaviour, morphology, neurobiology etc. There is also a large cosmid collection covering almost the whole C. elegans genome, which is used to determine the complete genomic sequence of the worm.
These characteristics of C. elegans make it the organism of choice for use as a tool in the drug discovery process. In particular, C. elegans may be used in the development of high throughput live animal compound screens, useful in the development of potential candidate drugs, in which worms are exposed to the compound under test and any resultant phenotypic and/or behavioural changes are recorded. The present inventors have developed a number of C. elegans-based screening methods which may be used to identify compounds which modulate the activity of SERCA, either directly or via the SERCA/PLB interaction. Compounds identified as modulators of SERCA activity using these screening methods may be useful as pharmaceuticals in the treatment of the wide range of diseases with which the SERCA genes have been associated.
Accordingly, in a first aspect the invention provides a method of identifying compounds which are capable of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:
contacting C. elegans which exhibit reduced SERCA ATPase activity compared to wild type C. elegans in one or more cell types or tissues with a compound under test; and
detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activity in the one or more cell types or tissues which exhibit reduced SERCA activity in the absence of the compound.
The method of the invention, which will be hereinafter referred to as the xe2x80x98up-regulation assayxe2x80x99 is performed using a C. elegans strain which exhibits reduced SERCA ATPase activity in one or more cell types or tissues, as compared to the SERCA ATPase activity in wild-type C. elegans. It has been observed that worms which exhibit reduced SERCA activity compared to wild-type worms manifest a variety of phenotypic and behavioural defects. The basis of the up-regulation assay is therefore to take worms which exhibit defects due to reduced SERCA activity, contact these worms with the compound under test and screen for phenotypic, behavioural or biochemical changes indicating a reversion towards wild-type SERCA activity. For example, worms with reduced SERCA activity often show a reduction in the rate of pharynx pumping. In this case, screening for an increase in the rate of pharynx pumping in the presence of a test compound would indicate a reversion towards wild-type SERCA activity due to the ability of the compound to enhance or up-regulate SERCA. For comparison purposes, an example of a C. elegans strain which exhibits xe2x80x98wild-typexe2x80x99 SERCA activity is the N2 strain (this strain can be obtained from CGC, University of Minn., USA). The N2 strain has been particularly well characterised in the literature with respect to properties such as pharynx pumping rate, growth rate and egg laying capacity (see Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern biological analysis of an organism, ed. by Henry F. Epstein and Diane C. Shakes, 1995 Academic Press; The nematode Caenorhabditis elegans, ed. by William Wood and the community of C. elegans researchers., 1988, Cold Spring Harbor Laboratory Press; C. elegans II, ed. by Donald L. Riddle, Thomas Blumenthal, Barbara J. Meyer and James R. Priess, 1997, Cold Spring Harbor Laboratory Press.).
C. elegans which exhibit reduced SERCA activity in one or more cell types or tissues can be obtained in several different ways. In a first embodiment, worms with reduced SERCA activity are obtained by treating a culture of worms with a chemical inhibitor of SERCA such as, for example, thapsigargin. As will be demonstrated in the examples given herein, treatment of C. elegans with thapsigargin results in recognisable phenotypic and behavioural changes such as paleness, reduced growth, pharynx pumping defects and production of very few progeny which are sick and grow very slowly. Accordingly, reversion of any one of these characteristics towards wild-type can provide an indication of a reversion towards wild-type SERCA activity.
In another embodiment, worms with reduced SERCA activity can be produced by specifically down-regulating the expression of SERCA in one or more tissues using antisense techniques or double stranded RNA inhibition. This can be achieved by transfection of C. elegans with a vector that expresses either an antisense C. elegans SERCA RNA or double stranded C. elegans SERCA RNA. Specific down-regulation of SERCA expression in different cell types or tissues of the worms can be achieved by incorporating into the vector an appropriate tissue-specific promoter to drive expression of the antisense RNA or double stranded RNA in the required tissues. SERCA expression will be specifically down-regulated only in those tissues which express the antisense RNA or double stranded RNA. By way of example, the promoter region of the C. elegans SERCA gene itself (see the examples given below) can be used to direct expression of an antisense RNA or double stranded RNA in all the cells and tissues which express SERCA. The C. elegans myo-2 promoter can be used to direct expression in the pharynx. The C. elegans myo-3 promoter can be used to direct expression in the body wall muscles. The use of antisense and double stranded RNA inhibition will be further understood with reference to the Examples included herein.
Alternative RNAi techniques which may be used to inhibit SERCA activity are described in the applicant""s co-pending International patent application No. WO 00/01846. These techniques, which are based on delivery of dsRNA to C. elegans by feeding with an appropriate dsRNA or feeding with food organisms which express an appropriate dsRNA, may lead to a more stable RNAi phenotype than results from injection of dsRNA.
In a still further embodiment, the C. elegans exhibiting reduced SERCA ATPase activity in one or more cell types or tissues may be a mutant strain in which SERCA activity is reduced but not eliminated i.e. a reduction-of-function mutant. The mutation may give rise to reduced SERCA activity through a down-regulation of SERCA expression in one or more cell types or tissues or through a defect in the SERCA protein itself or a defect in regulation of the activity of the SERCA protein.
A reduction-of-function mutant or a knock-out mutant can be isolated using a classical non-complementation screen, starting with a heterozygote C. elegans strain carrying a mutant SERCA allele on one chromosome and a recessive marker close to the wild-type SERCA allele on the other chromosome. The worms are subjected to mutagenesis using standard techniques (EMS or UV-TMP are suitable for this purpose) and the progeny is screened by eye for defects, especially in tissues which express SERCA. Since the screening is performed in the F1 generation, mutations will only give rise to a phenotype if the mutation occurs in the SERCA gene (due to non-complementation) or if the mutation is dominant, which does not occur frequently. These two possibilities can be distinguished in subsequent generations. A newly introduced SERCA mutation should be linked to the recessive marker. As a further control, DNA sequencing can be performed to determine the nature of the mutation.
The step of xe2x80x98detecting a phenotypic, biochemical or behavioural change in the C. elegans indicating a reversion towards wild type SERCA activityxe2x80x99 may be performed in several different ways. The method of choice is generally dependent upon the phenotype/behavioural characteristics of the starting worm strain, which is in turn generally dependent upon the nature of the cell types or tissues in which SERCA activity is reduced.
Inhibition experiments, for example the RNAi experiments and thapsigargin experiments described herein, demonstrate that SERCA is a vital protein for C. elegans. Moreover, reduction of SERCA activity results in a variety of phenotypes that can be used as basis of an assay to isolate compounds that alter the activity of SERCA. The main defects, and hence phenotypes, associated with reduced SERCA activity are related to muscle function e.g pharyngeal muscle, body wall muscle, vulva muscle, anal repressor muscle, and anal sphincter muscle. Screens based on reversion of defects in these muscles to wild-type can be used to identify compounds and genes that alter the activity of SERCA. Moreover, other phenotypes, such as paleness, reduced growth, reduced progeny, protruding vulva and protruding rectum can be used to identify compounds and genes that alter the function of SERCA.
In one embodiment, particularly suitable for use when the starting worm strain exhibits defects in pharynx pumping due to reduced SERCA activity in the pharynx (as compared to wild-type C. elegans) the up-regulation assay can be based on detection of changes in the pharynx pumping efficiency. If the starting worm strain exhibits a reduced rate of pharynx pumping due to reduced SERCA activity in the pharynx, then an increase in the rate of pharynx pumping in the presence of a test compound can be used as an indicator of a reversion towards wild-type SERCA activity in the pharynx.
C. elegans feeds by taking in liquid containing its food (e.g. bacteria). It then spits out the liquid, crushes the food particles and intemalises them into the gut lumen. This process is performed by the muscles of the pharynx. The process of taking up of liquid and subsequently spitting it out, requiring contraction and relaxation of muscles, is called pharyngeal pumping or pharynx pumping.
Alterations in SERCA activity influence the pharyngeal pumping rate. In particular, inhibition of SERCA using thapsigargin causes a reduction in the rate of pharynx pumping. Measurement of the pumping rate of the C. elegans pharynx is hence a method to determine the activity of SERCA. The pharynx pumping efficiency can be conveniently measured by placing the nematodes in liquid containing a fluorescent marker molecule precursor, such as calcein-AM. Calcein-AM present in the medium is taken up by the nematodes and the AM moiety is cleaved off by the action of esterases present in the C. elegans gut, resulting in the production of the fluorescent molecule calcein. As the quantity of calcein-AM that is delivered in the gut is dependent of the pumping rate of the pharynx, and hence of the activity of SERCA, the fluorescence measured in the gut of the formed calcein is a quantitative and qualitative measurement of the SERCA activity. It would be readily apparent to one skilled in the art that other types of marker molecule precursor which are cleavable by an enzyme present in the gut of C. elegans to generate a detectable marker molecule could be used instead of calcein-AM with equivalent effect.
In further embodiments, particularly suitable for use when the starting worm strain exhibits reduced SERCA activity in the vulva muscles, the up-regulation assay can be based on detection of changes in the egg laying behaviour of the C. elegans or on detecting changes in the amount of progeny produced by the C. elegans. 
Defects associated with reduced SERCA activity in the vulva muscles include defects in the production and laying of eggs and hence a reduction in the number of progeny produced. Typically, worms with reduced SERCA expression in the vulva are not able to lay their eggs. The eggs thus hatch inside the mother, which then dies. These mothers are easy to recognize under the dissection microscope. As a consequence of the egg laying defect, these worms produce less progeny, and hence the culture as a whole grows much more slowly. Defects associated with reduced SERCA activity have also been observed in the gonad, including the sheath cells and the spermatheca. These defects also result in reduced egg formation and hence a reduced egg laying phenotype.
One convenient way in which the egg production and egg laying behaviour of the worms can be monitored is by counting the number of resultant offspring produced. A variety of different techniques can be used for this purpose. For example, the offspring can be measured directly using the growth rate assay and/or the movement assay described below. Alternatively, specific antibodies and fluorescent antibodies can be used to detect the offspring. Any specific antibody that only recognizes eggs, or L1 or L2 or L3 or L4 stage worms, will only recognize offspring, such a specific antibody that recognizes an antigen on the L1 surface has been described by Donkin and Politz, W13G 10(2):71. Finally, the number of eggs or offspring in each well can be counted directly using a FANS device. The FANS device is a xe2x80x98worm dispenser apparatusxe2x80x99 having properties analogous to flow cytometers such as fluorescence activated cell scanning and sorting devices (FACS) and is commercially available from Union Biometrica, Inc, Somerville, Mass., USA. The FANS device, also designated a nematode flow meter, can be the nematode FACS analogue, described as fluorescence activated nematode scanning and sorting device (FANS). The FANS device enables the measurement of nematode properties, such as size, optical density, fluorescence, and luminescence and the sorting of worms based on these properties.
In a still further embodiment, particularly suitable for use when the starting worm strain exhibits reduced SERCA activity in the anal sphincter or the anal repressor, the up-regulation assay can be based on detection of a change in the defecation behaviour of the C. elegans. 
A reduction in the SERCA activity in the anal sphincter and/or the anal repressor, for example following treatment with thapsigargin, results in worms which are constipated and also in worms with a protruding rectum. Changes in the defecation rate of the worms can therefore also serve as an indicator of SERCA activity.
Defecation rate can be measured using an assay similar to that described above for the measurement of pharynx pumping efficiency, but using a marker molecule which is sensitive to pH. A suitable marker is the fluorescent marker BCECF. This marker molecule can be loaded into the C. elegans gut in the form of the precursor BCECF-AM which itself is not fluorescent. If BCECF-AM is added to worms growing in liquid medium the worms will take up the compound which is then cleaved by the esterases present in the C. elegans gut to release BCECF. BCECF fluorescence is sensitive to pH and under the relatively low pH conditions in the gut of C. elegans (pH less than 6) the compound exhibits no or very low fluorescence. As a result of the defecation process the BCECF is expelled into the medium which has a higher pH than the C. elegans gut and the BCECF is therefore fluorescent. The level of BCECF fluorescence in the medium (measured using a fluorimeter on settings Ex/Em=485/550) is therefore an indicator of the rate of defecation of the nematodes.
Defecation can also be measured using a method based on the luminescent features of the chelation of terbium by aspirin. The method requires two pre-loading steps, first the wells of a multi-well plate are pre-loaded with aspirin (prior to the addition of the nematode worms) and second, bacteria or other nematode food source particles are pre-loaded with terbium using standard techniques known in the art. C. elegans are then placed in the wells pre-loaded with aspirin and are fed with the bacteria pre-loaded with terbium.
The terbium present in the pre-loaded bacteria added to the wells will result in a low level of background luminescence. When the bacteria are eaten by the nematodes the bacterial contents will be digested but the terbium will be defecated back into the medium. The free terbium will then be chelated by the aspirin which was pre-loaded into the wells resulting in measurable luminescence. The luminescence thus observed is therefore an indicator of nematode defecation.
It has been observed that a reduction in SERCA activity, for example using inhibition by thapsigargin or double stranded RNA inhibition, results in a reduction in the growth rate of a C. elegans culture. Growth rate of the culture as a whole is reduced because the worms produce fewer progeny and also because the few progeny that are produced show poor/delayed growth. Cultures of worms which produce many healthy progeny grow faster than cultures of worms with few and/or sick progeny. Hence measurement of the growth rate of a culture of C. elegans is in indication of the activity of SERCA in the individual worms of the culture.
Growth rate can be monitored by measuring the number of eggs or the number offspring present in the culture, by measuring the total fluorescence in the culture (this can be autoflourescence, or fluorescence caused by a transgene encoding a flourescent or luminescent protein), but can also be measured using the movement screen described below. Alternatively, the growth rate of a culture of C. elegans can also be assayed by measuring the turbidity of the culture. In order to perform this xe2x80x98turbidity assayxe2x80x99 the worms are grown in liquid culture in the presence of E. coli or other suitable bacterial food source. As the culture of worms grows the food source bacteria will be consumed. The greater the number of worms in the culture, the more food source bacteria will be digested. Hence, measurement of the turbidity or optical density of the liquid culture will provide an indirect indication of the number of worms in the culture. By taking sequential measurements over a period of time it is possible to monitor the growth rate of the whole C. elegans culture.
As an alternative to the above-described methods, the growth rate and amount of progeny can be measured on a plate. Slow growing nematodes, nematodes with vulva defects and nematodes with gonad defects will produce less progeny within a certain time compared to nematodes which do not have these defects. Preferentially, the amount of offspring produced is scored on day five and on day eight. In experiments where the amount of offspring is reduced very drastically due to severe defects in the vulva, gonad or growth rate reduction, the offspring can also be scored at later time intervals.
In a still further embodiment, the up-regulation assay can be performed by detecting changes in the movement behaviour of C. elegans. As is illustrated by the examples included herein, SERCA is widely expressed in the muscles of C. elegans, including the muscles of the body wall. A reduction of SERCA activity in the body wall muscles gives rise to worms with movement defects. These strains can be used as the basis of an assay in which the worms are contacted with a compound under test and any changes in the movement behaviour of the worms are observed. Compounds which cause the defective movement to revert towards wild-type movement behaviour are scored as compounds capable of enhancing/up-regulating the activity of SERCA.
Changes in the movement behaviour of the worms can obviously be detected by visual inspection, but as an alternative a number of non-visual approaches for analysing the movement behaviour of worms have been developed which can be performed in a multi-well plate format and are therefore suitable for use in high-throughput screening. Nematode worms that are placed in liquid culture will move in such a way that they maintain a more or less even (or homogeneous) distribution throughout the culture. Nematode worms that are defective in movement will precipitate to the bottom in liquid culture. Due to this characteristic of nematode worms as result of their movement phenotype, it is possible to monitor and detect the difference between nematode worms that move and nematodes that do not move. Advanced multi-well plate readers are able to detect sub-regions of the wells of multi-well plates. By using these plate readers it is possible to take measurements in selected areas of the surface of the wells of the multi-well plates. If the area of measurement is centralized, so that only the middle of the well is measured, a difference in nematode autofluorescence (fluorescence which occurs in the absence of any external marker molecule) can be observed in the wells containing a liquid culture of nematodes that move normally as compared to wells containing a liquid culture of nematodes that are defective for movement. For the wells containing the nematodes that move normally, a low level of autofluorescence will be observed, whilst a high level of autofluorescence can be observed in the wells that contain the nematodes that are defective in movement.
In an adaptation of the movement assay, autofluorescence measurements can be taken in two areas of the surface of the well, one measurement in the centre of the well, and on measurement on the edge of the well. Comparing the two measurements gives analogous results as in the case if only the centre of the well is measured but the additional measurement of the edge of the well results in an extra control and somewhat more distinct results.
As an alternative to the above-described embodiments of the up-regulation assay which are all based on the observation of changes in phenotypic and/or behavioural characteristics of the C. elegans as an indicator of SERCA activity, the inventors have developed a method of analysing SERCA activity in a given cell type or tissue which is based upon the use of the marker molecule apoaequorin which is sensitive to changes in intracellular Ca2+.
Aequorin is a calcium-sensitive bioluminescent protein from the jellyfish Aequorea victoria. Recombinant apoaequorin, which is luminescent in the presence of calcium but not in the absence of calcium, is most useful in determining intracellular calcium concentrations and even calcium concentrations in sub-cellular compartments. Expression vectors suitable for expressing recombinant apoaequorin and, in addition, vectors expressing apoaequorin proteins which are targeted to different sub-cellular compartments, for example the nucleus, the mitochondria or the endoplasmic reticulum are available commercially (see below).
As SERCA is a endoplasmic reticulum-localized calcium pump, an apoaequorin that is targeted to the endoplasmic reticulum (hereinafter referred to as erAEQ) is particularly useful for developing assays for SERCA activity. Such apoaequorin is available from Molecular probes (Eugene, Oreg., USA). The vector erAEQ/pcDNAI (Molecular Probes) contains an Ig 2b heavy chain gene from mouse, an HA1 epitope and a recombinant apoaequorin in fusion. The mouse gene targets the aequorin to the endoplasmic reticulum, and the aequorin is mutated to make it less sensitive to calcium, as the concentrations of this ion are relatively high in the endoplasmic reticulum. Although apoaequorin is the calcium sensor of choice, it would be apparent to persons skilled in the art that any other calcium sensor localized in the endoplasmic reticulum could be used with equivalent effect
Plasmid expression vectors which drive expression of the ER-localized apoaequorin in C. elegans can be easily constructed by cloning nucleic acid encoding erAEQ downstream of a promoter capable of directing gene expression in one or more tissues or cell types of C. elegans, such that the promoter and the erAEQ-encoding sequence are operatively linked. As used herein the term xe2x80x9coperatively linkedxe2x80x9d refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. In a typical cloning procedure, the apoaequorin gene in fusion with the signals to locate the resulting protein to the endoplasmic reticulum was isolated from erAEQ/pcDNAI by EcoRI digestion and cloned into pBlue2SK. The erAEQ was then isolated as an EcoRI/Acc65I fragment by partial digestion and cloned in the vector pGK13 digested with the same enzymes.
Suitable promoters include the pharynx-specific promoter myo-2, the C. elegans SERCA promoter which directs expression in a wide range of muscle tissues and the body wall muscle-specific promoter myo-3. The vectors can then be used to construct transgenic C. elegans according to the standard protocols known to those of ordinary skill in the art. Expression of erAEQ allows for the determination of the calcium levels in the endoplasmic reticulum of various C. elegans cells and tissues, using the protocols of the manufacturer of erAEQ, or minor modifications thereof. Alterations in SERCA activity influence the concentration of calcium in the endoplasmic reticulum as SERCA functions as an endoplasmic reticulum calcium pump. Hence the apoaequorin luminescence measured in the assay is directly related to SERCA activity.
The basic xe2x80x98up-regulation assayxe2x80x99 methodology can also be adapted to perform a genetic screen in order to identify C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of SERCA. Accordingly, the invention also provides a method of identifying C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:
subjecting a population of C. elegans with wild-type SERCA activity to random mutagenesis;
allowing the mutagenized C. elegans to grow for one or two generations;
treating the mutagenized C. elegans to reduce the activity of the SERCA ATPase in one or more cell types or tissues; and
scoring a phenotypic, biochemical or behavioural characteristic of the C. elegans as an indicator of SERCA ATPase activity in the C. elegans in the said one or more cell types or tissues.
This genetic screen differs from the xe2x80x98up-regulationxe2x80x99 assay used to identify compounds in that the C. elegans are subjected to a random mutagenesis step before they are treated to reduce the activity of the SERCA ATPase. The random mutagenesis step can be performed using any of the techniques known in the art. EMS and UV-TMP mutagenesis, both of which are well known in the art (see Methods in Cell biology Vol. 48, 1995, ed. by Epstein and Shakes, Academic press) are preferred. After mutagenesis the worms are grown for one or two generations before they are treated to reduce the activity of SERCA. After one generation, the worms are heterozygous for any mutation, after two generations they may be homozygous or heterozygous for any mutation. Therefore growth for one generation leads to isolation of dominantly acting suppressors, growth for two generations yields both recessively and dominantly acting suppressors.
The step of treating the C. elegans to reduce the activity of the SERCA ATPase preferably comprises either treating the worms with a chemical inhibitor of SERCA, for example thapsigargin, or specifically down-regulating the expression of SERCA using antisense or double-stranded RNA inhibition.
When thapsigargin is added to worms in plate or liquid culture few progeny are produced and these don""t grow as well as wild-type worms. To perform a genetic screen based on thapsigargin inhibition wild-type worms are first subjected to standard mutagenesis protocols (using EMS or UV/TMP or any other mutagen). F1 or F2 progeny of the mutagenized worms are distributed individually to standard growth medium with bacteria, to which 10 to 50 mM thapsigargin is added. After 4-8 days the cultures are inspected for growth of progeny, either by eye or using the xe2x80x98turbidity assayxe2x80x99, as described above. Wild-type C. elegans with an integrated transgenic array causing general expression of a reporter protein such as GFP can also be used. In this case, cultures are inspected for growth of progeny either by eye or by detecting expression of the reporter protein.
Thapsigargin causes a short term pharynx pumping defect. Hence, the genetic screen can also be performed by measuring changes in the pharynx pumping efficiency. Wild-type worms are mutagenized and grown on solid media according to standard techniques known in the art. Adults are washed off the plates and put in buffer with calcein-AM and thapsigargin (an assay buffer of 40 mM NaCl, 6 mM KCI, 1 mM CaCl2, 1 mM MgCl2 can be used for this purpose). After two hours the worms are viewed under a fluorescence microscope and individual worms that show far brighter gut fluorescence than the other worms are selected, placed individually onto fresh plates and grown for an additional generation. Calcein-AM uptake in the presence of thapsigargin is then re-checked.
Inhibition of SERCA by antisense or double stranded-RNA inhibition will result in the same phenotypes as described above for the up-regulation assay and hence the same screens can be used to select for mutants that enhance or up-regulate SERCA activity. The precise nature of the screen used depends on the tissue in which the antisense or double stranded SERCA RNA is expressed.
An analogous genetic screen can also be performed using a reduction-of-function mutant C. elegans strain which exhibits reduced C. elegans activity in one or more cell types or tissues. Accordingly, in a further aspect the invention provides a method of identifying C. elegans which carry a mutation having the effect of enhancing or up-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises the steps of:
subjecting a population of mutant C. elegans which exhibit reduced SERCA activity in one or more cell types or tissues to random mutagenesis;
allowing the mutagenized C. elegans to grow for one or two generations; and
scoring a phenotypic, biochemical or behavioural characteristic of the C. elegans as an indicator of SERCA ATPase activity in the C. elegans in the said one or more cell types or tissues.
A suitable reduction-of-function mutant strain can be isolated as described above.
The basis of the above-described genetic screens is to screen for mutations that have the effect of enhancing or up-regulating SERCA activity and thus suppress the inhibitory effect of thapsigargin treatment, antisense or double stranded RNA inhibition of SERCA expression or a reduction-of-function mutation. Mutations likely to be identified using the method of the invention include mutations in genes involved in transcription and/or translation of SERCA, mutations that influence Ca2+ cycling between the ER and cytoplasm, mutations that influence Ca2+ buffering and mutations that influence the activity of Ca2+ binding proteins. Once a mutant worm has been identified using a genetic screen it is a matter of routine to identify the mutated gene using techniques commonly used in the art.
In summary, the up-regulation assay which may be used to identify compounds which enhance the activity and/or expression of SERCA is based on the use of C. elegans worms in which the activity or expression of the C. elegans SERCA protein is reduced. This may be achieved in at least three different ways. First, mutants can be selected that show reduced SERCA activity. Second, wild-type, mutant, or transgenic C. elegans strains can be treated with compounds that inhibit SERCA activity, such as thapsigargin. Third, RNAi technology can be applied to wild-type, mutant or transgenic C. elegans to reduce the SERCA activity. In each case, screening can be performed to select for compounds that enhance SERCA activity. Such screens may be based on the pharynx pumping rate, egg laying or movement. In a particular example of the up-regulation assay, wild-type, mutant or transgenic strains can be made transgenic for apoaequorin or another calcium marker. These markers may be expressed in the various tissues, such as the pharynx, the body wall muscles, the oviduct, vulva-muscles etc, for which specific promoters are known in the art. Apoaequorin may also be expressed more generally in C. elegans, for instance under the control of the SERCA promoter. The apoaequorin may further be fused to a specific signal peptide translocating the apoaequorin to the endoplasmic reticulum. Selecting compounds that enhance the activity or the expression of SERCA will enhance calcium uptake, and hence increase the bio-luminescence of the apoaequorin located in the lumen of the endoplasmic or sarcoplasmic reticulum.
In a second aspect the invention provides a method of identifying compounds which modulate the interaction between a sarco/endoplasmic reticulum calcium ATPase and phospholamban, which method comprises:
exposing transgenic C. elegans which contains a first transgene comprising nucleic acid encoding a vertebrate PLB protein and which expresses a SERCA protein to a compound under test; and
detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating an increase in the activity of the SERCA protein.
The vertebrate phospholamban (PLB) protein used in this second method of the invention, hereinafter referred to as the xe2x80x98interaction assayxe2x80x99 can be any vertebrate PLB protein. Most preferred are pig PLB (GenBank P07473) or human PLB (GenBank P26678) or a humanized pig PLB (see below). Mutant PLB proteins which exhibit stronger or weaker inhibition of SERCA relative to the wild-type protein may also be used.
The SERCA protein expressed by the C. elegans may be a C. elegans SERCA protein, a vertebrate SERCA protein, a fusion between a vertebrate SERCA protein and C. elegans SERCA protein or a mutant SERCA protein, for example a mutant which exhibits greater sensitivity to PLB.
The vertebrate SERCA protein can be any vertebrate SERCA isoform. Preferred isoforms are pig SERCA2a (GenBank P11606), human SERCA1a (GenBank AAB 53113), human SERCA1b (GenBank AAB 53112), human SERCA2a (GenBank P16614) and human SERCA2b (GenBank P16615). Human and pig SERCA2a are most preferred.
Various types of fusion proteins between C. elegans SERCA and vertebrate SERCA proteins which may be used in the method of the invention are described in the accompanying Examples. For example, the fusion might comprise the N-terminal part of C. elegans SERCA and the C-terminal part of a vertebrate SERCA.
It is essential that a xe2x80x98functionalxe2x80x99 combination of SERCA and PLB is chosen i.e. that the SERCA protein and the PLB protein are able to interact with each other such that the activity of SERCA can be inhibited by the PLB, mimicking the regulatory interaction occurring in vertebrates.
In the context of this application the term xe2x80x9ctransgenexe2x80x9d refers to a DNA construct comprising a promoter operatively linked to a protein-encoding DNA fragment. The construct may contain additional DNA sequences in addition to those specified above. The transgene may, for example, form part of a plasmid vector. By the term xe2x80x9coperatively linkedxe2x80x9d it is to be understood that the promoter is positioned to drive transcription of the protein-encoding DNA fragment.
Methods of preparing transgenic C. elegans, including worms carrying multiple transgenes, are well known in the art and are particularly described by Craig Mello and Andrew Fire, Methods in Cell Biology, Vol 48, Ed. H. F. Epsein and D. C. Shakes, Academic Press, pages 452-480. A typical approach involves the construction of a plasmid-based expression vector in which a protein-encoding DNA of interest is cloned downstream of a promoter having the appropriate tissue or cell-type specificity. The plasmid vector is then introduced into C. elegans of the appropriate genetic background, for example using microinjection. In order to facilitate the selection of transgenic C. elegans a second plasmid carrying a selectable marker may be co-injected with the experimental plasmid.
The plasmid vector is maintained in cells of the transgenic C. elegans in the form of an extrachromosomal array. Although plasmid vectors are relatively stable as extrachromosomal arrays they can alternatively be stably integrated into the C. elegans genome using standard technology, for example, using gamma ray-induced integration of extrachromosomal arrays (methods in Cell Biology, Vol 48 page 425-480).
The DNA fragment encoding the SERCA protein or the PLB protein may be a fragment of genomic DNA or cDNA. Preferably the DNA encoding the vertebrate SERCA protein is operatively linked to the promoter region of a SERCA gene. Most preferably the promoter region of the C. elegans SERCA gene is used. The term xe2x80x98promoter regionxe2x80x99 as used herein refers to a fragment of the upstream region of a given gene which is capable of directing a pattern of gene expression substantially identical to the natural pattern of expression of the given gene.
Provided that a functional combination is chosen, wherever the SERCA protein and the vertebrate PLB are co-expressed the two proteins will interact such that PLB inhibits the activity of SERCA. The aim of the interaction assay is to identify compounds which directly or indirectly disrupt the SERCA/PLB interaction, leading to an increase in SERCA activity. The increase in SERCA activity is monitored indirectly, by detecting phenotypic, biochemical or behavioural changes in the C. elegans which are indicative of an increase in SERCA activity. Advantageously, the nucleic acid encoding PLB is operatively linked to a tissue-specific promoter. With the use of a promoter of appropriate specificity, the vertebrate PLB can be expressed in all the cells of C. elegans, in a given type of tissue (i.e. all muscles), in a single organ or tissue (for example, the pharynx or the vulva), in a subset of cell types, in a single cell type or even in a single cell.
By restricting the expression of PLB to certain tissues it is possible to specifically down-regulate SERCA activity in these tissues and thus to influence the phenotype of the resultant transgenic worms. For example, when PLB is expressed in the pharynx, the resultant inhibition of SERCA activity in the pharynx results in a reduction in the rate of pharynx pumping. When PLB is expressed in the vulva muscles, the resultant inhibition of SERCA activity in the vulva results in an egg laying defect.
Although the interaction assay may be performed using functional combinations of C. elegans SERCA (especially mutant versions thereof, as discussed below) and vertebrate PLB, it is preferred to use functional combinations of vertebrate SERCA and vertebrate PLB. In order to ensure that the interaction assay can be used to identify compounds which specifically modulate the vertebrate SERCA/vertebrate PLB interaction it is preferred to use a transgenic strain which has been modified such that expression of the endogenous C. elegans SERCA protein is abolished or substantially reduced down to background levels. This may be achieved by introducing the transgenes encoding the vertebrate SERCA and PLB into a mutant strain having a knock-out or loss-of-function mutation in the chromosomal C. elegans SERCA gene (e.g. strain ok190 described in the accompanying Examples). A protocol for isolating a suitable knock-out mutant strain is given in the examples included herein. In a variation of this approach, expression of the endogenous C. elegans SERCA gene may be abolished/reduced using RNAi technology, as described hereinbefore. In this case, the genetic background of the transgenic C. elegans may be wild-type.
In a further embodiment, a vertebrate-specific interaction assay may be achieved by using transgenic C. elegans expressing a mutant version of the vertebrate SERCA protein which is resistant to a chemical inhibitor of SERCA activity, such as thapsigargin. The mutation Phe259Val renders C. elegans SERCA resistant to inhibition with thapsigargin. Equivalent mutations may be introduced into transgenes encoding the vertebrate SERCA proteins using standard site-directed mutagenesis. Applying the SERCA inhibitor, e.g. thapsigargin, to transgenic C. elegans which express a resistant mutant vertebrate SERCA and a vertebrate PLB will result in inhibition of the endogenous C. elegans SERCA only. Thus, if the inhibitor is added to the interaction assay in addition to the test compound, the screen will be specific for the interaction between the vertebrate SERCA and the vertebrate PLB.
A particular variant of the interaction assay uses a mutant version of the C. elegans SERCA protein which is more sensitive to vertebrate PLB proteins, such as, for example, a C. elegans SERCA containing the KDDKPV (SEQ ID NO:39) insertion. As illustrated in the accompanying Example 9, introduction of the amino acid sequence KDDKPV (SEQ ID NO:39) into the C. elegans SERCA protein results in a more efficient interaction between the mutant SERCA and vertebrate PLB. Therefore, double transgenic C. elegans strains containing a first transgene encoding a vertebrate PLB protein and a second transgene encoding a C. elegans SERCA KDDKPV (SEQ ID NO:39) insertion mutant may be used in the interaction assay.
In order to provide specificity for the mutant SERCA/PLB interaction, it is preferred that the double transgenic is also modified such that expression of the endogenous C. elegans SERCA gene is abolished or substantially reduced. As described above, this may be achieved by using a mutant C. elegans genetic background having a knock-out or loss-of-function mutation in the chromosomal SERCA gene or by using RNAi technology to inhibit SERCA expression. Alternatively, it is possible to engineer the mutant SERCA so that in addition to the KDDKPV (SEQ ID NO:39) insertion it also carries a firther mutation which renders it resistant to a SERCA inhibitor other than PLB, e.g. the thapsigargin resistance mutation Phe259Val. Addition of the SERCA inhibitor, e.g. thapsigargin, to the assay will result in specific inhibition of the endogenous C. elegans SERCA protein but not the resistant mutant.
As with the xe2x80x98up-regulation assayxe2x80x99 described above, the step of xe2x80x9cdetecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating an increase in the activity of SERCAxe2x80x9d can be performed in several different ways.
In one embodiment, particularly suitable for use when the transgenic C. elegans expresses PLB in the pharynx, the method is performed by detecting changes in the pharynx pumping efficiency. The rate of pharynx pumping can be measured using a marker molecule precursor such as calcein-AM, as described above for the up-regulation assay.
In still further embodiments, particularly suitable for use when the transgenic C. elegans expresses PLB in the vulva, the method can be performed by detecting changes in the egg laying behaviour of the C. elegans or by detecting changes in the number of progeny produced by the C. elegans. The number of progeny produced by the C. elegans can, as described above in connection with the up-regulation assay, be directly counted or can be measured indirectly using a growth assay or a turbidity assay.
In a still further embodiment, again particularly suitable for use when the transgenic C. elegans expresses PLB in the pharynx, SERCA activity in cells of the C. elegans pharynx can be monitored using apoaequorin luminescence. To achieve this the C. elegans are transfected with a third transgene which comprises nucleic acid encoding an apoaequorin protein, preferably ER-targeted apoaequorin, operatively linked to promoter capable of directing gene expression in the C. elegans pharynx. The construction of suitable expression vectors comprising such a transgene has been described hereinbefore.
In summary, the basic SERCA-PLB interaction screen to select for compounds that inhibit the interaction between SERCA and PLB is based on the construction of transgenic C. elegans expressing PLB. The PLB may be of any vertebrate origin, such as human or pig. The PLB may be expressed ubiquitously or in specific tissues, such as the pharynx, the body wall muscles, the oviduct, vulva muscles etc, for which specific promoters are known in the art. Preferred configurations of the interaction assay are summarised below, however, this is not intended to be limiting to the scope of the invention:
Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a vertebrate SERCA; expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,
Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a fusion between C. elegans SERCA and a vertebrate SERCA; expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,
Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a mutant vertebrate SERCA which is resistant to a SERCA inhibitor other than PLB, e.g. thapsigargin; wild-type genetic background; inhibitor is added to the assay in addition to the compound under test to specifically inhibit endogenous C. elegans SERCA expression,
Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a mutant C. elegans SERCA which is more sensitive to inhibition by ivertebrate PLB (e.g. KDDKPV (SEQ ID NO:39) insertion); expression of endogenous C. elegans SERCA abolished/reduced by mutation of the SERCA gene in the genetic background or by using RNAi on wild-type genetic background,
Double transgenic C. elegans, first transgene encoding a vertebrate PLB, second transgene encoding a double mutant C. elegans SERCA which is (i) more sensitive to inhibition by vertebrate PLB (e.g. KDDKPV (SEQ ID NO:39) insertion) and (ii) resistant to inhibition by a SERCA inhibitor such as thapsigargin (e.g. Phe259Val); wild-type genetic background; inhibitor is added to the assay in addition to the compound under test to specifically inhibit endogenous C. elegans SERCA expression.
In a third aspect the invention provides a method of identifying compounds capable of down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises:
exposing transgenic C. elegans containing a transgene comprising nucleic acid encoding a SERCA protein operatively linked to a promoter capable of directing gene expression to a sample of the compound under test; and
detecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a decrease in the activity of the SERCA protein.
The SERCA protein used in this third aspect of the invention, hereinafter referred to as the xe2x80x98down-regulation assayxe2x80x99 can be any SERCA isoform from any species. Preferably the SERCA protein is C. elegans SERCA, pig SERCA2a, or a human SERCA isoform, most preferably human SERCA 2A.
Preferably the nucleic acid encoding the SERCA protein is operatively linked to a tissue-specific promoter. Most preferably, the tissue-specific promoter is the C. elegans myo-2 promoter which directs tissue-specific expression in the pharynx.
In a preferred embodiment the transgenic C. elegans further contain a second transgene comprising nucleic acid encoding a reporter protein operatively linked to a promoter which is capable of directing gene expression in one or more cell types or tissues of C. elegans. The reporter protein is preferably an autonomous fluorescent protein, for example, a green fluorescent protein or a blue fluorescent protein or a luminescent protein.
Transgenic C. elegans over-expressing SERCA are generally observed to be starved and show delayed growth. Compounds which reduce or down-regulate the activity of SERCA will cause a reversion or reduction of this phenotype towards a wild-type phenotype. Accordingly, these worms can be used as a basis of a screen to identify compounds capable of reducing or down-regulating the activity of SERCA, by bringing the worms into contact with the compound under test and then detecting a reversion of the over-expression phenotype reflecting a decrease in the activity of the SERCA transgene.
The step of xe2x80x9cdetecting a phenotypic, biochemical or behavioural change in the transgenic C. elegans indicating a decrease in the activity if the SERCA proteinxe2x80x9d can be performed in several different ways. As mentioned above, transgenic C. elegans which overexpress the SERCA protein exhibit delayed growth. Accordingly, it is possible to look for a reversion of the overexpression phenotype by comparing the growth rate of the transgenic C. elegans in the presence and the absence of the compound under test. Compounds which increase the growth rate of the C. elegans culture are scored as compounds which are capable of reducing or down-regulating SERCA activity. Any of the growth assay methods described in connection with the xe2x80x98up-regulationxe2x80x99 assay could be used for this purpose.
Transgenic C. elegans which overexpress SERCA also exhibit altered egg laying behaviour and reduced pharynx pumping. Hence, the down-regulation assay can also be performed by detecting changes in the egg laying behaviour or the pharynx pumping efficiency, as described previously.
In summary, the basic down-regulation assay consists of introducing extra SERCA into C. elegans and screening for a compound that inhibits SERCA activity. The SERCA introduced into C. elegans may be C. elegans SERCA or a SERCA of any vertebrate origin, such as human or pig. The SERCA protein may be expressed ubiquitously or in specific tissues such as the pharynx, the body wall muscles, the oviduct, the vulva muscles etc, for which appropriate tissue or cell type-specific promoters are known in the art.
The above-described methodology for the down-regulation assay can be adapted to perform a genetic screen to identify C. elegans carrying a mutation having the effect of reducing or down-regulating SERCA activity. Thus, in a further aspect the invention provides a method of identifying C. elegans which carry a mutation having the effect of reducing or down-regulating the activity of a sarco/endoplasmic reticulum calcium ATPase, which method comprises the steps of:
providing a transgenic C. elegans strain containing a first transgene comprising nucleic acid encoding a SERCA protein operatively linked to a promoter capable of directing gene expression in one or more cell types or tissues of C. elegans; 
subjecting a population of the said C. elegans strain to random mutagenesis;
allowing the mutagenized C. elegans to grow for one or more generations; and
scoring a phenotypic, biochemical or behavioural characteristic of the C. elegans as an indicator of SERCA ATPase activity in the C. elegans in the said one or more cell types or tissues.
The genetic screen is preferably carried out using transgenic C. elegans containing an integrated transgene harboring 20-50 ng/xcexcl pGK7 (containing the C. elegans genomic SERCA gene, including the promoter region, see examples given below) and a general GFP expressing construct. These worms are starved and show general growth delay. The same results are obtained using a vertebrate SERCA, such as the human or pig SERCA.
Alternatively, the screen can be performed using transgenic nematodes containing an integrated transgene harboring the genomic C. elegans SERCA gene operatively linked to the myo-2 promoter, and a general GFP expressing construct. These worms are also starved and show growth delay.
The worms are grown and subjected to random mutagenesis according to standard techniques known in the art. The mutagenized worms then are distributed individually to standard growth medium with supplemented with food source bacteria. After 4-8 days the cultures are inspected for growth of progeny, either by eye, by using any of the growth assay techniques mentioned previously in connection with the up-regulation assay, using the turbidity assay or by counting the numbers of progeny produced.
The basis of these genetic screens is that mutations having the effect of reducing or down-regulating SERCA activity will suppress the effect of SERCA over-expression. Mutations identified using this screen may include mutations in genes involved in transcription and/or translation of SERCA, mutations that influence Ca2+ cycling between the ER and the cytoplasm, mutations that influence Ca2+ buffering and mutations that influence the activity of Ca2+ binding proteins.
In the field of human pharmaceuticals, compounds identified as modulators of SERCA activity using the screening methods of the invention may be useful leads in the development of pharmaceuticals for the treatment of the wide range of diseases with which the SERCA genes have been associated, such as cardiac hypertrophy, heart failure, hypertension, NIDDM, Darier-White disease, Brody""s disease.
Outside the pharmaceutical field, compounds identified as modulators of SERCA activity may find important applications as pesticides, particularly insecticides, herbicides or nematocides. Maintaining high calcium concentrations in the ER is important for the proper synthesis of protein, including translation, folding, glycosylation, processing and transport. Treatment of living organisms with chemicals that inhibit the activity of SERCA will hence have a negative effect on the welfare of these organisms. As such, SERCA inhibitors are potential pesticides or can be considered as basic compounds for the development of pesticides such as herbicides, insecticides and nematocides. It has been shown that SERCA function is essential in the intracellular trafficking of the Notch receptor in drosophila (Periz et al., 1999 EMBO J; 5983-5993). This studies and others indicate that SERCA is an interesting target for pesticidal intervention. Accordingly, the screening methods described herein could be applied to screen for pesticides.